The use of our models
1. Which size (small or large) should I order?
It depends of the test protocol: large cultures allow easiest application of small quantities of creams (2 mg/cm² for example); moreover, MTT assay and histology analysis can be performed on the same culture, and more medium is available for analysis (cytokine measurement, ...)
2. If I order the reconstituted human epidermis (RHE) at day 10 or 12, can I use the same medium than the one used for the cultures at day 17?
Yes, the medium is the same, but growth medium have to be used for more than 48 hours of culture.
3. What I have to do when I receive the tissues?
The SkinEthic human tissue models should be removed from the agarose-nutrient solution in the shipping multiwell plate immediately after arrival in the laboratory, according to the guidelines below.
Upon arrival, remove the multiwell plate from the aluminium bag and strip off the white tape. Open the plate under a sterile airflow and remove the sterile filter paper. Carefully take out each insert (or HTS plate) containing the tissue, rapidly remove any remaining agarose that adheres to the outer sides of the insert by gentle blotting on the sterile filter paper, and immediately place in a plate in which each well has previously been filled with an appropriate volume of SkinEthic Maintenance or Growth Medium (at room temperature)(+ make sure that the medium is never above the polycarbonate filter).
Act quickly as the tissue cultures dry out rapidly when not in contact with medium. Make sure that no air bubbles are formed underneath the insert!
Place culture in the incubator at 37°C, 5% CO2, and saturated humidity.
4. How can I apply the test products?
It depends of the test product: powder, liquid, cream, gel...
|Nature of test product||Volume or quantity (RHE/S)||Application method|
|Hydrosoluble (liquid)||Minimal volume: 5 µl||Micropipette + nylon mesh* + spread with an inoculating loop|
|100 µl||Micropipette + spread with an inoculating loop|
|Oils||Minimal volume: 1 µl||Positive displacement pipette** + spread with an inoculating loop|
|Creams, gels||1 µl to 4 µl||Positive displacement pipette + spread with an inoculating loop|
|5 µl||Positive displacement pipette + spread with an paint brush n°2***|
|Solids||According protocol||Applied with syringe and spread with an inoculating loop|
*Mesh (Nylon 150 µm, ref: 03150/44)
Mesh is prepared with Biopsy punch d = 8 mm
** Microman, Gilson.
***Raphael, n°2 petit gris n° 835.
5. I want to perform a permeability assay: can I replace the maintenance medium by any other solution underneath the cultures?
No, the maintenance medium has to be used. If an other solution is used, a preliminar cytotoxity test has to be performed.
6. How can I detach reconstituted human epidermis from the polycarbonate filter?
Dispase Protocol is used to detach the cultures from the filter:
- Put the insert (which has little feet) with the epidermis on 1 to 2 ml of Dispase II solution at room temperature.
- After 3-5 minutes, the epidermis will come off the polycarbonate and can be lifted with forceps. You then need to rinse the epidermis by spreading it on a small amount of medium on which it will float.
- Product information on Dispase grade II: Boehringer Mannheim, cat. number 295 825. (solution at 2.4 U/ml).
7. Can we obtained the EpiSkin model in other stade of culture than 13 days?
The EpiSkin model can be provide at 6 days of culture. In this case no controls at day6 can be provided to the users. Only controls at day 13 are performed and demonstrate a good differenciation of the EpiSkin model in normal conditions.
8. Is the RHE model validated for corrosivity?
STATEMENT ON THE APPLICATION OF THE SKINETHIC™ HUMAN SKIN MODEL FOR SKIN CORROSIVITY TESTING
At its 25th Meeting, held on 16-17 November 2006 at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy, the non-Commission members of the ECVAM Scientific Advisory Committee (ESAC)1 unanimously endorsed the following statement: On the basis of a peer review2 of the results of an inter-laboratory study3 with the SkinEthic™ reconstituted human epidermal (RHE) model, the Committee endorses the conclusion that the SkinEthic™ human skin model can be used for distinguishing between corrosive and non-corrosive chemicals within the context of the OECD test guideline, TG 431.
Head of Unit ECVAM
Institute for Health & Consumer Protection Joint Research Centre European Commission
Assessment of the human epidermis model SkinEthic RHEfor invitro skin corrosion testing of chemicals accordingto new OECD TG 431
Helena Kandárová(a), Manfred Liebsch(a), Horst Spielmann(a),Elke Genschow(a), Elisabeth Schmidt(a), Dieter Traue(a), Robert Guest(b),Andrew Whittingham(b), Neil Warren(b), Armin O. Gamer(c), Marina Remmele(c),Tanja Kaufmann(c), Elke Wittmer(c), Bart De Wever(d), Martin Rosdy(d)
a ZEBET, Bundesinstitut für Risikobewertung, BfR, Diedersdorfer Weg 1, D-12277 Berlin, Germany
b Safepharm Laboratories, Derby, UK
c Experimental Toxicology and Ecology of BASF AG, Ludwigshafen, Germany
d SkinEthic Laboratories, Nice, France
Received 2 June 2005; accepted 21 November 2005.
9. Is the RHE model validated for skin irritation of chemicals?
At its 29th meeting, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the following statement “It is concluded that the performance of SkinEthic RHE assay in an external catch up validation study (MTT end point), met the criteria outlined to be considered to have sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non skin irritating) test substances.”(*)
This statement reinforces the SkinEthic leadership in tissue engineering with two validated methods: EpiSkin in 2007 and RHE in 2008.
The SkinEthic RHE validated protocol, 42 min exposure, 42 hours incubation time, based on a simple cytotoxic end-point (MTT), showed a good specificity (80%) and sensitivity (90%), a low False positive rate (20%) and False negative rate (10%) with an overall accuracy of 85% during the validation process by three lead and participating laboratories.
SkinEthic patented RHE model is a fully differentiated reconstructed epidermis in a chemically defined medium and manufactured according to strict quality controls during mass production process. This ready-to-use model, also validated for skin corrosion, is exclusively commercialized by SkinEthic Laboratories, an ISO 9001 certified company.
RHE and the others patented models proposed worldwide by SkinEthic will reduce the animal use in cosmetics and chemicals testing. They are available to Industries, Contract Research Organizations and Academic Research Institutions that promote in vitro toxicology testing.
(*)Statement of the scientific validity of in-vitro tests for skin irritation testing, ECVAM, November, 5th, 2008, officially released December, 3rd, 2008.
10. Is the EpiSkin model validated for corrosivity?
The EPISKIN model is scientifically validated for corrovivity assessment (TIV 12, 483-524, 1997, Inscription at the Annex V of Directive for Dangerous Substances Publication at the J.O. European Community Commission Directive 2000/33/EC and Directive 86/609/EC Guide line OCDE 431.
11. Is the EpiSkin model validated for skin irritation of chemicals?
Clichy, 30th April 2007 - ECVAM (European Centre for the Validation of Alternative Methods) has announced the validation of an ‘in vitro’ test as a full replacement method for assessing the skin-irritancy potential of chemicals. This test employs the L’Oréal Episkin model; a human epidermis reconstructed on collagen.
“More than twenty years of research on skin reconstruction have been recognized today” said Jean-François Grollier, Executive Vice-President Research and Development at L’Oréal. “It is a great step towards the elimination of animal use.”
Since the eighties, L’Oréal has been working to develop reconstructed human skin to better understand its biological behaviour, as well as being able, in the future, to assess the tolerance of ingredients and finished products without animal tests.
This work has so far contributed to L’Oréal’s ability to end animal tests on all finished products in 1989. “Today, Episkin is routinely used for tolerance evaluation of our products and our commitment to develop alternative methods continues”, added JF Grollier.
The validation of Episkin is a particularly important result as the skin-irritancy potential of chemicals is today carried out on animals. Importantly, the test will also enable the replacement of animal tests for skin-irritancy of 10,000 substances, as foreseen under the implementation of REACH.
According to ECVAM, Episkin ‘predicts the skin irritancy potential of chemicals with great accuracy and precision and will therefore fully replace tests’ on animals.
12. Do we freeze / stick in fridge the tissues on arrival?
Always leave them at room temperature.
13. How the MTT solution is prepared for viability assay?
A stock solution of MTT powder (Sigma Cat No, M5655) at 5 mg/ml is prepared in PBS, filtered and stored at -20°C in 1 ml samples.
The day of the experiment, a sample is diluted at 10% in SkinEthic Maintenance medium.
14. How can I fix the cultures for histology analysis?
Carefully cut the polycarbonate filter out of its plastic ring with a sharp scalpel.
Fill a 1.5 ml vial with buffered formaldedhyde 4% (W/V) ( ex:Sigma ref HT50-1-1 ; Carlo Erba Ref N°526932) and put the sample in it.
The sample must not dry (at any step of the procedure).
15. Which phototype of the reconstructed human pigmented epidermis should I order for my experiment?
For whitenning tests, we recommend to use phototype VI and phototype IV for pigmentation induced tests.
Ethical and regulatory framework
1. What is REACh?
REACH is the acronym for Registration, Evaluation and Authorisation of CHemicals. The REACH proposal requires industry to register all existing and future new substances with a new European Chemicals Agency.
Existing substances (about 30,000) have to be registered within the first 11 years, the so-called phase-in period. The Commission's proposal basically prioritises these substances based on produced or imported annual volume.
All substances will have to go through one or more stages of the REACH process as described below.
• For all substances produced or imported in quantities of 1 ton or more per year, manufacturers and importers must prepare a registration dossier to be submitted to the European Chemicals Agency. It is industry’s task to gather and assess the required information (requirements mainly based on volume, comprises data on physicochemical, toxicological and eco-toxicological properties). In addition to these data on the substance, individual identified uses of downstream users throughout the supply chain as well as assessments of the associated risks and safety measures derived from these must be specified. If further testing needs to be undertaken a test plan is required as well.
• For substances with annual volumes of more than 10 tons, the assessment of the safe handling (Chemical Safety Assessment) must be documented in a Chemical Safety Report.
• Dossier evaluation: The Member States authorities can check the compliance of any registration dossier with the requirements of REACH, and examine and endorse the testing proposals provided by the industry.
• Substance evaluation: The Member State authorities are allowed to examine registration dossiers in order to evaluate whether a substance presents a risk to human health or the environment, and to determine the need for possible authorisation or restriction of marketing and use.
• Authorisation will be required for each use of a substance belonging to specific groups, i.e. substances of very high concern - CMRs category 1 and 2 (carcinogenic, mutagenic or toxic to reproduction), PBTs (persistent, bio-accumulative and toxic), vPvBs (very persistent and very bio-accumulative) and other substances identified as causing serious and irreversible effects on humans and the environment.
• Authorisation will be granted for these uses if the manufacturer or importer is able to demonstrate that risks can be adequately controlled. If such evidence cannot be provided, authorisation can only be granted if an analysis shows that the socio-economic advantages of the specific use are predominant.
• If a risk is identified as not being adequately controlled, a proposal to restrict marketing and use of a substance can be made by the Commission or a Member State. The decisions on restriction are taken by the Commission in consultation with the Member States.
The new European Chemicals Agency will be established to facilitate the registration tasks (including establishing and maintaining the necessary databases). The Member States still have the responsibility for evaluation and authorisation phases. The final decision on authorisation will be taken by the Commission, in consultation with the Member States.
2. What are the 3R?
Founded in 1959, this forms the basis of the ethical process applied to animal testing in Europe and North America.
After graduating the suffering of animals used in experiments in English laboratories, W.M.S. Russell and R.L. Burch developed a program for setting up and developing "humane" guidelines, called the "3 R's rule" including the following points:
- Reduce the number of animals being tested
- Refine the methodology used, this implies the notion of end-points)
- Replace the animal models
This concept has gradually been adopted by various institutions to establish lines for conducting animal experimentation: the Canadian Council on Animal Care, American Animal welfare Act and British Government (Home Office). It has been introduced into law by:
The Council of Europe (convention STE N° 123)
The European Union (Directive n° 86/609/EEC)
and France (decree no. 87-848; decrees of 19 April 1988); 10 measures seeking to establish a real animal experimentation policy in public research organizations (decree no. 2001-464; decree no. 2001-486) .
This objective, initially good practices in animal experimentation, has, since Russell and Burch, been introduced into law. It features as a recommendation in the preamble of the convention STE123 and Directive 86/609/EEC as well as in their translation into French law (decree 2001-486).
Using fewer animals in experiments may be achieved through different methods:
Limitation to only those experiments considered as absolutely necessary.
Reduction of useless repetitions. This does not mean going against one of the fundamental principles of experimentation which requires the confirmation of original results by independent peers, but rather avoiding the repetition of previous studies conducted in another country, and seeking to meet national or Community legislative/regulatory requirements (Directive no. 86/609/EEC) in view of a marketing authorization or innocuousness/toxicity verification. Efforts to harmonize safety regulations on the European/international level (e.g. toxicological tests, etc.), to approximate standards in force (protection of animal and human health and the environment) and to achieve mutual recognition of the validity of protocols and scientific data will reduce the number of studies required by various State authorities and therefore the number of animals used.
Drafting of an experimental protocol before any experiment, in the idea that a well-prepared study is highly likely to provide conclusive results and thus it will be unnecessary to verify through other tests on a new series of animals. This may result in:
The elimination of debatable investigations. It is under an ethics committee remit to put forward improvements to the experimental protocols whose objectives are unclear, procedures are not detailed enough, animal models are unsuitable or the number of animals inadequate, and to reject the protocol if the applicant does not agree to apply the improvements requested.
Use of statistics in the drawing up of the experimental protocol for a preliminary estimation of the number of animals required for obtaining results that are statistically useable.
Sharing of animals in experiments between colleagues conducting research, either related but with different techniques or different but compatible.
Use of homogeneous animals in terms of biological characters and, amongst other things, health status, so as to reduce the variability of individuals from each other as far as possible.
Maximum exploitation of data obtained during an experiment.
While it is important to organize necessary experiments in such a way that they produce exploitable results with the least amount of animals possible, this reduction effort should nonetheless avoid a situation where less reliable results are obtained.
The concept of refining, in other words optimizing experiments, is based on the methodology applied to animals with a view to:
Reducing, eliminating or relieving discomfort, pain, distress or anguish suffered by the animals,
Obtaining more pertinent information at the lowest possible cost about animals' "ill-being".
This endeavor to refine must continue throughout an animal's lifetime and during the various stages of the experimental protocol.
Refining before experimentation involves:
Carefully choosing the animal model used by taking account of the current state of knowledge, this concerns both the pertinence of the choice of species and the protocol itself,
Improving the transportation, breeding and housing conditions (care, enrichment of the environment) of the animals with a view to increasing their well-being as far as possible,
Correctly planning the protocol so as to avoid any disturbances likely to
limit the experiment validity (animals stressed by overcrowding in the housing facility or by entry into the protocol without time to acclimatize for example),
end up with the failure to finish the whole experiment in optimum conditions (availability of the experiment site, equipment, etc.).
establishing the earliest end-points possible on the basis of the experiment's objective. In this regard, one of the most positive roles of the regional ethics committee for animal experimentation will be to advise on evaluating the "cost/benefit" balance of the project.
Refining during the experiment itself concerns the methods chosen and the procedures used. At each possible moment, it should be planned to:
- give preference to non-invasive procedures (imagery, in vivo, telemetry),
- give appropriate pre, per and post-experiment care,
- resort to anesthesia/analgesia,
- avoid painful tests (Draize test, skin irritation test, use of stimulants) when replacement methods exist,
- reduce the length of certain studies, especially toxicological studies,
- study acute rather than chronic situations,
- apply the end-points established beforehand,
- use appropriate euthanasia procedures.
Refining after the experiment means optimum use of the results obtained (e.g. statistical studies).
This petition of principle encourages researchers to replace as often as possible the model in vivo by models in vitro or "in silico" (mathematical, bioinformatic models). It may be applied in a number of areas, particularly safety studies in the broad sense, provided that the model used has been duly validated and is used in the scope of its validation.
However, it is clear that as knowledge currently stands, these methods are usually complementary to a global project when, in themselves, they do not constitute a research objective.
There is great pressure exerted by law and the public to encourage the development of these so-called "alternative" methods that can constitute real substitutes in certain areas of fundamental and applied research.
Refining, according to W.M.S. Russell and R.L. Burch, means, among other things, doing everything possible to avoid/limit the pain/suffering endured by animals. In this regard, it is up to the personnel conducting the experiments to fix end-points.
According to the CCPA (Canadian Council on Animal Care), the end-point is defined "as the point at which an animal's pain and/or distress is terminated, minimized or reduced by taking actions such as: killing the animal humanely; terminating a painful procedure; or giving treatment to relieve pain and/or distress".
Objective of end-points
The establishment of end-points aim to avoid:
death or morbid state as an end-point, as was often the case in the past (toxicology, infectious diseases, cancerology),
unnecessary pain/suffering. When the pain/suffering endured by an animal exceeds a threshold fixed beforehand, the decision is made to end it,
incorrect data obtained from stressed or suffering animals.
Establishment of end-points
Before embarking on experiments, it is essential to fix end-points, which falls successively back on:
- anticipating the emergence of pain, regardless of the planned experimental protocol, but all the more so when the pain is foreseeable (production of monoclonal antibodies, toxicology studies or in cancerology, invasive procedures, etc.)
- fixing the end-points involves:
determining the most significant indicators of pain/suffering in the specific research circumstances:
- change in physical appearance/body weight/ingesta/behavior
- clinical signs
- change in biological parameters
- responses to external stimuli
subsequently choosing the earliest end-point compatible with the study's scientific objectives.
Establishing the conduct to maintain:
Clearly define the role of personnel involved (personnel conducting the experiments, vets, managers of the health state of animals, personnel working with regard to the animals' housing, care or treatment) so that everyone knows when and how to intervene.
Determine how often experimental animals will be observed. When the appearance of pain/suffering is foreseeable, this frequency must be increased.
Application of end-points
During an experiment, it is necessary to:
- Recognize pain,
- Quantify it according to a scale,
- Act once predefined end-points have been reached,
- Note in an animal register the observations made and steps taken.
3. What is the 7th amendment of cosmetic european directive?
The seventh amendment of the Cosmetics Directive, adopted in 2003, took account of the widespread public view that animal testing was undesirable for cosmetic products, although it appeared more justifiable for pharmaceuticals and other potentially toxic chemicals. The Directive provides for a ban on animal testing of finished products (applicable from 11 September 2004), and a complete ban on animal testing of cosmetic ingredients as soon as alternative methods are validated and adopted by EU legislation. The final deadline of 11 March 2009 applies even if alternative tests are not available then.
A marketing ban on animal-tested products also applies, from 11 September 2004 or as soon as alternative methods are validated and adopted and again by 11 March 2009 at the latest, with the exception of three study areas where development of alternatives will unavoidably take longer – alternative tests for repeated dose toxicity, reproductive toxicity and toxicokinetics need not be introduced until 11 March 2013.
But who decides if an alternative method guarantees the safety of a product, or offers assurance equivalent to that of traditional testing methods?
In the EU it is ECVAM, the European Centre for Validation of Alternative Methods, which is part of the EU’s Joint Research Centre in Ispra, Italy. Since 1992, ECVAM has focused on the development and validation of alternative testing methods, with the aim of replacing, reducing and refining the use of laboratory animals. Its work is on in vitro methods, using cell and tissue cultures, and in silico methods – computer modelling based on the relationship between chemical structure and biochemical, physiological and biokinetic modelling, known as quantitative structure activity relationships or QSAR.
Alternative methods developed and validated by ECVAM are then adopted in EU legislation, through the work of the Enterprise and Industry DG’s Cosmetics and Medical devices Unit, which takes the lead on developing policies across this sector(1). In addition to becoming EU law, the new methods are fed into the OECD and, if accepted, become international guidelines valid for the 30 OECD member countries and also many non-members.
ECVAM’s Dr Thomas Hartung explains: “Our final goal is that our validated tests become an OECD-accepted methodology. That is why we celebrated last year’s success when the first full replacements for animal testing were accepted at OECD level(2). Once tests are performed according to the OECD’s guidelines and its principles of good laboratory practice, the results have to be accepted by other countries. This mutual acceptance avoids the need for companies to repeat experiments for authorisation to market products abroad.”
The OECD itself is running only a limited number of validation studies on alternative methods, but works closely with the ECVAM and its American counterpart ICCVAM. Japan is also in the process of establishing a national validation centre, and it is hoped that the initial steps of Japanese researchers in this area will lead to further international coordination.
ECVAM is coordinating the efforts of companies developing alternative methods so that the EU timetable for replacement of animal methods can be met. Possible candidate tests for validation are selected from their developers, and prioritised. In some cases, ECVAM participates in the validation studies. Typically, the validation of a new test method takes three years and costs about $300,000. “Cosmetic companies are very actively involved through their association COLIPA,” Hartung continues. “Many are interested in participating at an early stage because they will be able to use these innovative methodologies for predictive testing, agent discovery and agent profiling even before the tests become mandatory. They promise higher throughput, lower costs and better public acceptance – and thus offer real market advantages.” Through the Commission’s Enterprise and Industry DG and with input from industry, ECVAM reports annually to the European Parliament and the Council of Ministers so that early warning can be given if progress slips behind the timetable.
Confidence in new technologies
An ad hoc group was set up – with representation from the cosmetics industry, animal welfare and consumer organisations, academia, the EU and the OECD – with the aims of reviewing the state of development of alternative methods in 11 key toxicological areas, and estimating the time needed for full replacement of animal testing for cosmetics:
Skin irritation and corrosion
Skin absorption and penetration
Subacute and subchronic toxicity
Genotoxicity and mutagenicity
UV-induced toxic effects
Toxicokinetics and metabolism
Reproductive and developmental toxicity
A very practical consequence of the development of alternative test methods is the emergence of an entirely new commercial service, providing facilities or take-away kits for in vitro eye irritation or skin sensitivity testing. Tests, and tissue-engineered materials which were developed originally for therapeutic purposes, are also being explored now for predictive testing.
Validation by ECVAM aims to maintain products’ safety level by demonstrating that these new alternative methods can do the job, whether in vitro, in silico or as a refined animal experiment.